Chae Hee-Don, Yun Jeanho, Bang Yung-Jue, Shin Deug Y
National Research Laboratory, Department of Microbiology, Dankook University College of Medicine, Cheonan 330-714, Korea.
Oncogene. 2004 May 20;23(23):4084-8. doi: 10.1038/sj.onc.1207482.
We previously reported that cdk2 phosphorylates two serine residues near the DNA-binding domain of the YA subunit of NF-Y transcription factor and this phosphorylation is essential for DNA binding of NF-Y. In this study, we examined the effects of a phosphorylation-deficient mutant form of YA, YA-aa, in which the two serine residues are replaced with alanine, on the cell cycle and expression of the NF-Y target genes. Transient transfection assays show that YA-aa inhibits transcription from the NF-Y target promoters, such as cdc2, cyclin A, and cdc25C. Moreover, this inhibitory function of YA-aa can be suppressed by the expression of wild-type YA, implying that YA-aa inhibits transcription of those NF-Y target genes by inactivating wild-type YA. Since NF-Y target genes include the cell cycle-regulatory genes that ensure orderly progression of the cell cycle, we examined the effects of YA-aa in cell cycle progression. We constructed a recombinant adenovirus encoding YA-aa and found that YA-aa expression leads to repression of cell cycle-regulatory genes, such as cyclin A, RNR R2, DNA polymerase alpha, cdc2, cyclin B, and cdc25C. Consistently, YA-aa expression results in the inactivation of both cdc2 and cdk2. Furthermore, cell cycle analysis reveals that YA-aa induces cell cycle arrest at both G1 and G2/M. These results suggest that cdk2-dependent phosphorylation of NF-Y is essential for the expression of the cell cycle-regulatory genes and therefore for cell cycle progression at both G1/S and G2/M.
我们之前报道过，细胞周期蛋白依赖性激酶2（cdk2）使核因子Y（NF-Y）转录因子的YA亚基DNA结合结构域附近的两个丝氨酸残基发生磷酸化，且这种磷酸化对于NF-Y与DNA的结合至关重要。在本研究中，我们检测了YA的磷酸化缺陷突变体形式YA-aa（其中两个丝氨酸残基被丙氨酸取代）对细胞周期以及NF-Y靶基因表达的影响。瞬时转染实验表明，YA-aa抑制来自NF-Y靶启动子的转录，如细胞周期蛋白依赖性激酶2（cdc2）、细胞周期蛋白A（cyclin A）和细胞周期蛋白磷酸酶25C（cdc25C）。此外，野生型YA的表达可抑制YA-aa的这种抑制功能，这意味着YA-aa通过使野生型YA失活来抑制那些NF-Y靶基因的转录。由于NF-Y靶基因包括确保细胞周期有序进行的细胞周期调控基因，我们检测了YA-aa对细胞周期进程的影响。我们构建了一种编码YA-aa的重组腺病毒，发现YA-aa的表达导致细胞周期调控基因如细胞周期蛋白A、核糖核苷酸还原酶大亚基R2（RNR R2）、DNA聚合酶α、细胞周期蛋白依赖性激酶2、细胞周期蛋白B和细胞周期蛋白磷酸酶25C的表达受到抑制。一致地，YA-aa的表达导致细胞周期蛋白依赖性激酶2和细胞周期蛋白依赖性激酶2均失活。此外，细胞周期分析表明，YA-aa在G1期和G2/M期均诱导细胞周期停滞。这些结果表明，NF-Y的cdk2依赖性磷酸化对于细胞周期调控基因的表达至关重要，因此对于G1/S期和G2/M期的细胞周期进程也至关重要。
